Bioflavonoid impregnated materials

ABSTRACT

Cellulosic fibrous materials are described which are impregnated with a bioflavonoid composition, the bioflavonoid content of the composition comprising at least naringin and neohesperidin. The use of such impregnated materials is also described, for example as paper or bamboo towels and cardboard, as well as the process for impregnating the materials.

FIELD

The present invention relates to bioflavonoid impregnated cellulosicfibrous materials, processes for impregnating the materials and theiruses. In particular, the invention relates to bioflavonoid impregnatedcellulosic fibrous materials such as paper, paper towels, bamboo fibreand cardboard and articles formed from such materials.

BACKGROUND

Cellulosic fibrous materials such as paper are used in a wide variety ofapplications, ranging from domestic use to commercial use in, forexample, hospitals, schools, kitchens and laboratories in the form of,for example, paper towels or face masks or even garments such as bamboofibre socks.

Some materials would benefit from having antimicrobial properties. Theseinclude for example, cardboard, paper, cleaning wipes, paper towels orface masks or even garments.

GB2468836 discloses compositions comprising bioflavonoid compounds andtheir antibacterial, antifungal and antiviral activity but no suggestionwas made that they could be used in impregnating fibres and materials.

SUMMARY OF THE INVENTION

The present invention relates to cellulosic materials impregnated with abioflavonoid composition.

According to a first aspect of the invention there is provided amaterial impregnated with a bioflavonoid composition, the bioflavonoidcontent of the composition comprising at least naringin andneohesperidin.

Especially preferred is when the major part of the bioflavonoid contentof the composition comprises naringin and neohesperidin. Preferably,naringin and neohesperidin together form at least 50% wt/wt, morepreferably at least 70% wt/wt, for example at least 75% wt/wt, forexample 75%-80% wt/wt of the bioflavonoid content of the composition(excluding other biomass).

The bioflavonoid content of the composition may further comprise one ormore compounds of Formula (I):

wherein R¹ is a hydroxyl or methoxyl and R² is hydrogen, hydroxyl ormethoxyl and X is hydrogen or a saccharide.

A preferred option is when R² is hydrogen and R¹ is in the 3- or4-position. Another option is when R¹ is 3-hydroxy and R² is 4-methoxyl.Preferably, X is H. More preferably, X is a saccharide.

In preferred embodiments, X is a disaccharide. Suitable disaccharidesinclude combinations of two monosaccharides, preferably pyranoses,linked by a glycosidic bond, for example rhamnose and glucose, forexample L-rhamnose and D-glucose.

Suitable disaccharides can have the structure:

wherein one of R³ and R⁴ is H and the other OH or both are H or both areOH. Preferably R³ is H and R⁴ is OH so that the disaccharide isrutinose.

Favoured aglycones of bioflavonoids for use in this invention are thedisaccharides 6-O-(alpha-L-rhamnopyranosyl)-beta-D-glucopyranose, alsoknown as rutinose, and2-O-(alpha-L-rhamnopyra-nosyl)-beta-D-glucopyra-rose.

Suitable compounds of Formula (I) include neoeriocitrin, isonaringin,hesperidin, neodiosmin, naringenin, poncirin and rhiofolin, in additionto naringin and neohesperidin. One of these compounds may be present inaddition to naringin and neohesperidin, although a mixture of two ormore of these compounds is particularly preferred.

Such mixtures can be obtained by extraction from bitter oranges and theend product is called citrus aurantium amara extract. Particularlypreferred are the mixtures of bioflavonoid obtained from the extract ofcrushed whole immature bitter oranges. The mixtures can also be derivedfrom the starting material comprised of the pith of immature, bitter(blood/red) oranges such as Seville oranges that are classed as‘inedible’ and from which the pips, flesh and oily skin have beensubstantially removed or remain undeveloped.

Suitable mixtures can include 2, 3, 4, 5, 6, 7, 8, 9 or more compoundsof Formula (I). A mixture comprising 2, 3, 4, 5, 6, 7, 8, or 9 of theabove named bioflavonoids is preferable, for example containing 3, orcontaining 4, or containing 5, or containing 6, or containing 7, orcontaining 8, or containing 9 of said bioflavonoids.

It is presently believed that mixtures of such bioflavonoids haveadvantages over the use of a single bioflavonoid. It is particularlyadvantageous that extract of bitter oranges is employed without the needfor isolating individual bioflavonoids. In an extract from bitteroranges biomass may be associated with up to 40-60% wt/wt, preferablyabout 55% wt/wt based on the weight of the bioflavonoid content of thecomposition. The biomass comprises pectins and other sugar derivedmaterials. If it is desired to avoid biomass, other solubilising agentssuch as dextrines, for example cyclodextrin, may be employed if desired.

A particular advantage of many compositions described herein is thatthey may employ compounds of natural origin. Thus, for example, it ispreferred to employ compounds of Formula (I) from bitter oranges.However synthetically or semi-synthetically obtained compounds may beemployed if desired instead of the ones directly extracted from naturalsources although this tends to be less favourable in view of cost.

The compositions may further comprise oleuropein. Preferably this isobtained from extraction from the leaf of the olive, for example Oleaeuropaea. Such extracts typically contain 5% to 80% wt/wt, morepreferably 10% to 70%, for example 20% wt/wt of oleuropein.

The wt/wt ratio of bioflavonoids to oleuropein can be 5:1 to 1:4,preferably 2:1 to 1:2, more preferably 1:2 to 1:1 and even morepreferably 3:2. In addition to the bioflavonoid content of thecomposition, the composition may further comprise one or more fruitacids, for example citric acid, malic acid, and ascorbic acid. One ormore of the acids are preferably neutralized with a suitable base, suchas a quaternary ammonium base, for example a choline base, such ascholine carbonate, bicarbonate or, preferably, hydroxide. Morepreferably, citric, malic and ascorbic acids are all used in thepreparation of the composition, and especially preferred is when theseare fully neutralized to provide citrate, malate and/or ascorbate salts.Especially preferred is choline ascorbate.

It has been found that the composition described herein is particularlyeffective in the presence of one or more organic acids. In oneembodiment, the composition further comprises one or more organic acids.

A surprisingly effective organic acid is salicylic acid or itspharmaceutically acceptable salt optionally together with a furtherorganic acid or pharmaceutically acceptable salt.

The salicylic acid may be obtained from willow bark extract.Alternatively, methods for synthesising salicylic acid are known tothose skilled in the art.

Sometimes it is preferred that the salicylic acid is in the form of theacid rather than its salt.

Similarly, a further organic acid if present is similarly in the form ofthe acid rather than its salt. Suitable further organic acids includeacids of up to 8 carbon atoms which are monobasic (i.e. one CO₂H group),di-basic or tri-basic acid which optionally contain 1, 2 or 3 hydroxylgroups. Such further organic acid may be one or more of citric acid,malic acid, latic acid, tartaric acid, fumaric acid and the like.

Such compositions can provide an approximately neutral or acid pH, whenused, for example from 3 to 8, more aptly 3.5 to 7, for example 4 to 5.

At present it is preferred to employ salicylic acid and citric acid inthe compositions.

Such compositions may include a solubilising agent, for example,salicylic acid such as a dextrin such as cyclodextrin.

The compositions described herein have an extremely favourable safetyand environmental profile. As well as showing extremely effectiveantimicrobial activity, the compositions are also non-toxic,non-corrosive, renewable and completely biodegradable. The compositionsdisclosed in WO 2012/017186 (herein incorporated by reference) are thepreferred compositions of the present invention.

The cellulosic fibrous materials of the invention may be composed ofpaper or cardboard or bamboo fibres. Paper is defined as a materialproduced from a cellulose pulp which may be derived from wood, rags orgrasses. The paper may be in the form of a paper towel, towelette,cloth, wipe or pad. Paper towels have a variety of applications, forexample, paper towels are used to dry a person's hands after washing,also known as hand towels. Paper towels or wipes are also used forcleaning purposes to wipe down surfaces in a hospital, laboratory or akitchen, for example, and can also be known as kitchen roll, kitchenpaper or kitchen wipes. Pads are cellulosic fibre sponges and haveapplication in personal hygiene and in medical kits. Wipes are producedas air-laid paper where the fibres are carried and formed to thestructure of paper by air.

The paper may be treated with softeners, lotions or added perfume tocreate a desirable “feel” or texture.

Bamboo materials may be formed of bamboo fibre which is a cellulosefibre extracted or fabricated from natural bamboo. Bamboo is asustainable crop and, as a natural product derived entirely from plantcellulose, bamboo fibre is biodegradable by microorganisms in soil andalso by sunlight. Preferably, the bamboo materials of the presentinvention are formed of 100% bamboo fibres although mixtures with othercellulose fibres are also contemplated.

The bamboo may also be in the form of a paper towel, towelette, wipe orpad which may have the same applications as paper towels. The bamboofibres may also be used as a clothing fabric, optionally in combinationwith other known fibres, to make garments, such as socks and hospitalgowns. For example, socks made from bamboo fibres impregnated with thebioflavonoid compositions described herein can help reduce foot odour.The bioflavonoid impregnated bamboo fibres are activated when they comeinto contact with moisture from the foot. For hospital gowns, thebioflavonoid composition is activated when the gowns come into contactwith, for example, blood or urine.

Fabrics made from bamboo fibres which are impregnated with thebioflavonoid compositions described herein are very useful in hospitalor care home environments. For example, the bamboo fabric can be usedfor bedding sheets, surgical drapes, curtains and the like where it isdesirable to use a material with antimicrobial properties.

Paper fibre fabrics can be used instead of the bamboo fibre fabricsdescribed herein; however, the bamboo fibre fabrics are preferred asthese fabrics are more durable than paper fibre fabrics.

Paper towels, bamboo towels and the like, may be heated, for example byusing a microwave, in order to provide a hot towel. These hot towels maybe disposable and/or re-heatable and can be used in restaurants, hotelsand on planes.

The bioflavonoid impregnated paper and/or bamboo fibres of the inventioncan also be provided in the form of a face mask, such as a respiratorymask or surgical mask, to provide the user with enhanced protectionagainst inhaling bacteria and viruses or to prevent or reduce the spreadof bacteria and viruses. The face masks may be reusable or disposable.Methods of manufacturing face masks are well known in the art.

Bioflavonoid impregnated bamboo and/or paper fibres can be used in theform of single or multi-ply food pads. Such food pads are often found inthe bottom of food packaging and can also be referred to as napkins orblankets. The use of these food pads is particularly desirable in foodpackaging containing food with a short shelf life, for example meat orfruit. The food product, for example, the meat or fruit generally sit ontop of the food pad within the packaging. The bioflavonoid impregnatedfood pad provides a dramatic reduction in the number of bacteria such asSalmonella, E. coli and Campylobacter which cause foods such as meat andfresh fruit to decay, reducing their shelf life. The bioflavonoidimpregnated food pads are particularly suitable in the packaging ofmeats, including poultry (e.g. chicken or turkey), lamb, beef and pork;fish, including salmon and prawns; and fruits including soft fruits suchas blackberries, raspberries, loganberries, strawberries and the like.

Cardboard is heavy duty paper and may include a single thick sheet ofpaper or more complex configurations such as multiple corrugated anduncorrugated layers which tend to by more durable than regular paper.The cardboard of the present invention will generally be of a depth ofless than about 1 cm. The impregnated cardboard can be used inpackaging, for example food packaging.

The cellulosic fibrous materials of the present invention are providedin a dry form and are activated when they are wetted, i.e. when thematerial comes into contact with moisture, such as a liquid. The liquidmay be, for example, water, body fluids, for example sweat, blood orurine, fruit juice, cooking juices and the like. The materials can bewetted before being applied to a surface to be cleaned, for example, byapplying water to the material before using on a surface. Alternatively,the materials are activated during use, for example, when drying handsmoisture is transferred onto the material or when using the material towipe down a wet surface.

The materials are provided in a substantially dry form and arepreferably dried by heating to constant mass.

Preferably, the amount of bioflavonoid coating impregnated in thematerial is uniform throughout the material.

The bioflavonoid compositions described herein are biodegradable and canbe impregnated into biodegradable materials such as biodegradable paper,bamboo fibres and the like to provide environmentally friendly products.

The bioflavonoid compositions described herein show activity against awide range of organisms including gram positive bacteria, gram negativebacteria, fungi, virus, protazoans and insect parasites. Thecompositions may be employed against difficult bacteria such asmethicillin resistant Staphylococcus aureus (MRSA), Clostridiumdifficile (C. diff), Helicobacter pylori (H. pylori), and vancomycinresistant enterobacteria. The compositions may also be used againstnorovirus and other pathogens whereby transmission is by contact on air.In particular, the compositions described herein show activity againstE. coli, S. aureus, Salmonella, B. subtilis and P. aeruginosa.

According to a second aspect of the invention, there is provided aprocess for impregnating the materials described herein with thebioflavonoid compositions described herein. Impregnation is the partialor total saturation of a material, although total saturation ispreferred. In particular the material is a thin material. A thinmaterial is defined as having a depth of less than about 1 cm.Impregnation may be after manufacture of the thin material or it mayoccur during manufacture of the thin material, for example, impregnationof the cellulose fibres before being formed into the material.

If impregnating pre-formed cellulosic fibrous material, the processinvolves immersing the material, in the bioflavonoid composition tototally or partially saturate the material with the composition. Thematerial may then be rolled, squeezed or wrung to remove any excess ofthe composition. The material is then dried, either by air dryingnaturally, oven drying or by mechanical drying. The equipment used tomechanically dry materials will be known to those skilled in the art aswill alternative drying methods. The process results in a dry materialwhich can then be packaged as desired and later activated by wetting.Alternatively, the cellulosic fibres used to produce the material mayfirst be immersed in the bioflavonoid composition to totally orpartially saturate the fibres with the composition which are then driedeither before or after being formed into materials such as paper orcardboard by methods known in the art.

Alternatively, the materials may be impregnated by spraying thebioflavonoid composition onto the materials so that the compositionimpregnates the outer surface region of the material to achieve at leastpartial impregnation. Spraying may also be used to impregnate the fibresduring manufacture or extraction, before being formed into the materialsof the invention.

A particular method for impregnating paper towels is disclosed inExample 3. Fibrous bamboo products may also be impregnated in the sameway as disclosed in Example 3.

Preferably, the processes described above provide uniform bioflavonoidimpregnation throughout the cellulosic fibrous material. A concentrationof between 0.005 and 0.75%, preferably between 0.005 and 0.5%, morepreferably between 0.025% and 0.5%, even more preferably between 0.025and 0.1% of the bioflavonoid composition is used. The compositionsdescribed herein are water soluble and water can be used to dilute thebioflavonoid composition to the desired concentration.

According to a third aspect of the invention, there is provided a methodof reducing the bacterial load on a surface. The method of reducing thebacterial load on a surface is provided by two mechanisms. Firstly, thekill is achieved by the action of the bioflavonoid compositions and thensecondly the contaminants are mechanically removed by the materialitself via the action of placing on and wiping the surface, i.e.mechanical wiping.

The surface may be any bioactive surface and could be either a human ornon-human surface. For example, a human surface may include the skin onthe hands, feet or face. A non-human surface may include any surface ofsanitary importance which may carry a contaminant, for example, thesurfaces found in schools, bathrooms, kitchens, factories, for examplefood factories, laboratories, hospitals and the like.

For food contact the Environmental Protection Agency (EPA) requires theactive to effect a 5 log reduction of the challenge organism in 30seconds. Preferably, the materials of the present invention effect atleast a 5 log reduction of the bacteria load on a surface in 30 seconds.

According to a fourth aspect of the invention, there is provided apackaged product wherein the product is formed of a dry cellulosicfibrous material impregnated with a bioflavonoid composition. Theproduct and the bioflavonoid composition are as described in the firstaspect of the invention.

The cellulosic product may be individually packaged. Alternatively, theproduct may be packaged as part of a multi-pack. Known packaging methodsand materials may be used to package the products of the presentinvention, for example conventional filmic agents or cardboard boxes.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more fully understood it will now bedescribed, by way of example only, and with reference to the followingFigure(s), in which:

FIG. 1 is a graph showing the results of the effects of differentdilutions of the Citrox BC active dried onto Bounty® brand paper towelson S. aureus activity.

FIG. 2 is a graph showing the results of the effects of differentdilutions of the Citrox BC active dried onto Bounty® brand paper towelson E. coli activity.

DETAILED DESCRIPTION

The bioflavonoid content may comprise 40-50%, for example about 45%wt/wt of the bioflavonoid composition. A suitable source of abioflavonoid composition is herein referred to as “HPLC 45” or “CitroxBC” of which about 45% (of the total composition of HPLC 45/Citrox BC)comprises bioflavonoids. The bioflavonoids are in admixture with biomassresidues of extraction from bitter oranges, such as pectins, sugars andminor organic acids, which make up the remaining 55%. HPLC 45 isavailable from Exquim (a company of Grupo Ferrer) as Citrus BioflavonoidComplex 45% HPLC.

TABLE 1 The mixture of bioflavonoids in HPLC 45 % bioflavonoid inmixture Bioflavonoid with biomass Neoeriocitrin 1.1 Isonaringin 1.2Naringin 23.4 Hesperidin 1.4 Neohesperidin 12.5 Neodiosmin 1.4Naringenin 1.5 Poncirin 2.0 Other (Rhiofolin) 0.5

EXAMPLES

Staphylococcus aureus was chosen as a representative gram positiveorganism. This organism is found on mammalian skin and is, therefore,shed into the surrounding environment. E. coli was chosen as therepresentative of the gram negative enteric bacteria. This organism isfound in the digestive tract of birds, mammals and reptiles. Itspresence in the environment signals fecal contamination. Pseudomonasaeruginosa was chosen to represent the non-enteric gram-negativebacteria. This genera of bacteria is present in water with relatedspecies representing major plant pathogens and human opportunisticpathogens. Bacillus subtilis was chosen as the representative grampositive spore-formers. This bacterium is found in soil and water but isalso ubiquitous in the environment. This species forms endospores as asurvival mechanism. Bacterial endospores are the most resistant form oflife on Earth and, therefore, represent an ongoing concern forsanitation, disinfection and sterilisation processes. Endosporesrepresent the “ultimate” challenge for any antimicrobial agent.

Example 1 Minimum Inhibitory Concentration (MIC) and MinimumBactericidal Concentration (MBC) Procedure

A pure culture of a single microorganism is grown in an appropriatebroth. The culture is standardized using standard microbiologicaltechniques to have a concentration of very near 1 million cells permillilitre. The more standard the microbial culture, the morereproducible the test results. The antimicrobial agent is diluted anumber of times, 1:1, using sterile diluents. After the antimicrobialagent has been diluted, a volume of the standardised inoculums equal tothe volume of the diluted antimicrobial agent is added to each dilutionvessel, bringing the microbial concentration to approximately 500,000cells per millilitre. The inoculated, serially diluted antimicrobialagent is incubated at an appropriate temperature for the test organismfor a pre-set period, usually 18 hours. After incubation, the series ofdilution vessels is observed for microbial growth, usually indicated byturbidity and/or a pellet of microorganisms in the bottom of the vessel.The last tube in the dilution series that does not demonstrate growthcorresponds with the minimum inhibitory concentration (MIC) of theantimicrobial agent.

In order to differentiate between a microbiostatic agent (bacteria arenot killed just inhibited) and a microbiocidal agent (bacteria arekilled) an MBC test is performed. When a microbiostatic agent is removedor neutralized, previously inhibited bacteria begin to grow again. Eachwell showing no growth/turbidity in the MIC test is sub-cultured onmedia that contains no biocide. Any microbial growth resulting from thistest indicates that, at that concentration, the active ismicrobiostatic. If the subculture results in no bacterial regrowth,then, at that concentration, the active is microbiosidal. The range ofconcentration of Citrox BC active tested was 0.075-0.75%.

Discussion of Results

The MIC test is an established “screen” for the biostatic (and possiblyalso biocidal) activity of liquid antimicrobials. It is often used tofind the appropriate concentrations of an antimicrobial active to usefor further efficacy testing. Performing both the MIC and MBC test willenable one to differentiate between a biocidal or biostatic mode ofaction. Depending on the concentration of active used and the contacttime an active will often demonstrate both biostatic and biocidal modesof action.

The range of Citrox BC active tested was 0.075%-0.75%. For P.aeruginosa, no MIC value was obtained as all concentrations of theCitrox BC active tested showed no turbidity (Table 2).

MCB testing showed that all concentrations were also bactericidal for B.subtilis, there was also no MIC value obtained demonstrating thatinhibition of growth took place at all concentrations tested. The MBCvalue obtained for B. subtilis was 0.315% Citrox BC active. This meansthat concentrations ranging from 0.075% to 0.315% are bacteristatic andall concentrations of the Citrox BC active greater than or equal to0.315% are bactericidal.

These results indicate that gram negatives like P. aeruginosa are moreeasily killed by the Citrox BC active than the gram positive B.subtilis.

TABLE 2 MIC/MBC Testing % MIC MBC Citrox (G/NG) (CFU/mL) BC P.a. B.s.P.a. B.s. 0 G 0 0 0 0.075 NG G 0 4.2 × 10² 0.095 NG G 0 3.1 × 10² 0.115NG G 0 3.3 × 10² 0.135 NG G 0 3.5 × 10² 0.155 NG G 0 3.6 × 10² 0.175 NGG 0 2.4 × 10² 0.195 NG G 0 1.5 × 10² 0.215 NG G 0 1.3 × 10² 0.235 NG G 01.6 × 10² 0.255 NG G 0 40 0.275 NG G 0 40 0.295 NG G 0 1 0.315 NG NG 0 00.335 NG NG 0 0 0.355 NG NG 0 0 0.375-0.750 NG NG 0 0 G = Growth, NG =No Growth P.a. = Psuedomonas aeruginosa B.s. = Bacillus subtilis

Example 2 Time Kill Test Procedure

All timed kill tests were performed using a standard viable countprocedure. Reference NB X34689.

The following neutralising solution was used in all kill tests.

Tween 80-3% Saponin-3% Histidine-0.1% Cysteine-0.1% Rationale

A timed kill test assesses the amount of time it takes to kill a definedpopulation of microorganisms. A wide variety of microorganisms arekilled by the Citrox BC active. An important first step incharacterising this active for use in an antimicrobial towel is toverify the kill claims. Claims for efficacy are based on the number ofbacterial killed within a defined time frame. The most rigorous claimsare those made for food contact where the active must affect a 5 logreduction of the challenge organism in 30 seconds.

Discussion of Results Example 2(a) Timed Kill Test: 10 Minute ContactTime

Bacterial kill kinetics are affected by bacterial numbers, theconcentration of active used and the contact time. In order to determinethe most effective range of the Citrox BC active, S. aureus was used ina 10 minute kill test to assess the efficacy of various concentrationsof the Citrox BC active. A >6.56 log reduction was observed for allconcentrations (0.45-0.65%) of the Citrox BC active tested (Table 3).

When B. subtilis was used as a challenge organism, 0.7% Citrox BC wasrequired to effect a >5 log reduction in 10 minutes (Table 4). Based onprevious tests, 0.5% active is the most effective for general use.

TABLE 3 Time Kill Test: S. aureus, 10 min. % Citrox Log10 Log BC CFU/mLCFU/mL Reduction 0 7.4 × 10⁶ 6.86 0 0.45 <2 0.3 6.56 0.5 <2 0.3 6.560.55 <2 0.3 6.56 0.6 <2 0.3 6.56 0.65 <2 0.3 6.56 0.65 + 6.6 × 10⁶ 6.810.05 neutralizer

TABLE 4 Time Kill Test: B. subtilis, 10 min. % Citrox Log10 Log BCCFU/mL CFU/mL Reduction 0 1.1 × 10⁶ 6.04 NA 0.5 2.9 × 10⁴ 4.4 1.64 0.7<2 0.3 5.74

Example 2(b) Timed Kill Test: 30 Second Contact Time

Timed kill studies using E. coli, P. aeruginosa and S. aureus wereperformed using 0.5% Citrox BC active with a contact time of 30 seconds.Log reductions of >6.4 were seen for all organisms (Table 5). Thisconfirms that this active would meet the criteria for use in foodcontact situations.

TABLE 5 Time Kill Test: 30 seconds E. coli P. aeruginosa S. aureus %Citrox BC 0 0.5 0 0.5 0 0.5 CFU/mL 5.1 × 10⁶ <2 6.4 × 10⁷ <2 7.4 × 10⁶<2 Log10   6.7 <0.3   7.8 <0.3   6.8 <0.3 CFU/mL Log NA 6.5 NA 7.3 NA6.5 Reduction

Example 2(c) Timed Kill Test: Sporicidal Activity

As stated above, the ultimate test for any antimicrobial active is theability to kill spores. Any chemical or process that kills a bacterialspore is, by definition, a sterilant. In order to assess if the CitroxBC active was sporicidal, a kill test was performed on an actual sporesuspension. Citrox BC, over a range 0.5% to 1.5%, was tested over a 1hour time period. There were some limitations to this test. The sporesuspension (B. subtilis, ATCC 6633, 6.4×10⁴ CFU/pellet, Microbiologics)in the test was only at ˜2×10⁴ CFU/ml, limiting the log reductioncalculation. The lyophilized pellets were found to contain charcoal, asubstance known to neutralise the bioflavonoid component of the CitroxBC active. With those limitations, approximately a 2 log reduction inspores was demonstrated. This indicates that the Citrox BC active hasdefinite activity against spores. Spore suspensions at a higher titerwithout a charcoal additive should be used to investigate this activityfurther.

Example 3 Surface Testing Using Paper Towel Impregnated with Citrox BCActive 1) Procedure: Adding Citrox BC to Paper Towel

Bounty® (“Bounty” is a registered trademark of Procter & Gamble) brandpaper towels were used to make the dry antimicrobial towels. Bounty®paper towels are a conventional, commercially available paper towelproduct. Citrox BC active concentrate was diluted to desiredconcentrations. One paper towel was immersed completely into the dilutedactive and then wrung out by hand. The towel was dried overnight.

2) Procedure: Weight of Citrox BC Active Dried onto Bounty® Brand PaperTowel

Bounty® brand paper towels were dried to a constant weight in a 54° C.oven. Various dilutions of the Citrox BC active were dried onto Bounty®brand paper towels as described above. The towels were dried at roomtemperature overnight. The treated towels were then dried to a constantweight at 54° C. The weight difference between the untreated and treatedtowels is presumed to be the weight of the Citrox BC active.

3) Procedure: For Testing Affect of Administration of Impregnated PaperTowels to a Surface

Using the lab bench top as a representative hard, non-porous surface, agrid was marked off using tape. Cotton-tipped swabs saturated with abroth culture of the challenge organism were used to inoculate thesurface and air dried. Paper towels treated with dilutions of the CitroxBC active were wetted and then used to clean the inoculated bench top.The bench top was visibly wet for 3 minutes (contact time) and thenallowed to completely air dry. RODAC (Replicate Organism Detection andCounting) plates were used to sample the cleaned surface for survivingbacteria. The plates were incubated overnight at a temperatureappropriate to the challenge organism. Colonies were counted and thenumber used to calculate CFU/cm². Results were calculated by averagingthe counts from five 3″×3″ “grid squares”.

4) Procedure: RODAC Sampling

A RODAC plate is used to touch the surface to be sampled after which theplate is incubated at an appropriate temperature. There are nutrients inthe media that promote the growth of a variety of microbes. Lecithin andPolysorbate 80 are incorporated in the agar and function asdisinfectant/sanitizer neutralisers. The type and number ofmicroorganisms is detected by the appearance of colonies on the surfaceof the agar medium. Collection of samples from the same area before andafter cleaning and treatment with a disinfectant permits the evaluationof sanitary procedures.

Results

Paper towels wetted with water and containing no Citrox BC active wereassessed for the ability to remove bacteria from a contaminated hardsurface. The results for this control (i.e. unimpregnated paper towels)are shown by the bar labelled “0” in FIGS. 1 and 2. FIGS. 1 and 2 showthe results for both S. aureus and E. coli. Paper towels containingdilutions of the Citrox BC active greater than 1:200 were able to reducethe levels of S. aureus from >50 CFU/cm² to <1 CFU/cm². The paper towelscontaining dilutions of the Citrox BC active greater than 1:200 wereable to reduce the levels of E. coli from >7 CFU/cm² to <1 CFU/cm².

These results show that a dry antimicrobial towel are activated bywetting.

Discussion of Results

Different dilutions of the Citrox BC active were dried onto Bounty®brand paper towels. These treated towels were used to decontaminate alab bench heavily inoculated with bacteria. The ability of the treatedtowels to affect a decrease of contaminants on the lab bench wasevaluated using RODAC plates.

A method was developed to assess ability of a paper towel impregnatedwith the Citrox BC active to reduce bacterial numbers on a contaminatedhard surface. RODAC plates are recommended for the detection andenumeration of microorganisms present on surfaces of sanitaryimportance. RODAC plates are specially constructed so that an agarmedium can be overfilled producing a dome-shaped surface that can bepressed on a surface for sampling its microbial content. RODAC platesare used in a variety of programs to establish and monitor cleaningtechniques and schedules.

When using a paper towel plus an antimicrobial active, one must keep inmind that removal of bacteria from a contaminated surface occurs by twomechanisms: first is the kill achieved by the action of theantimicrobial active and second is mechanical removal of thecontaminants by the paper towel itself.

Lab scale antibacterial towels were used to calculate the weight of theCitrox BC active dried onto the towels. The weight of active present onthe towel (Table 6) can be used as a starting point for cost analysis.

TABLE 6 Weight of Citrox BC active dried onto Bounty ® paper towel Post-Ave. wt. Citrox Pre- treatment of BC BC treatment Dry active DilutionDry Weight Weight Difference (g/towel) Std Dev 1:150 4.32547 4.31821−0.00726 0.00388 0.006875 4.31226 4.32089 0.00863 4.31476 4.324000.00924 4.32424 4.32607 0.00183 4.32625 4.33321 0.00696 1:100 4.308774.35320 0.04443 0.05244 0.010257 4.30530 4.36125 0.05595 4.32380 4.370210.04641 4.30800 4.35446 0.04646 4.30435 4.37329 0.06895 1:50  4.317394.49804 0.18065 0.20090 0.046566 4.38309 4.51604 0.13295 4.31517 4.541910.22674 4.31539 4.52472 0.20933 4.32305 4.57787 0.25482

1. A dry cellulosic fibrous material impregnated with a bioflavonoidcomposition, the bioflavonoid content of the composition comprising atleast naringin and neohesperidin.
 2. The material of claim 1, whereinthe naringin and neohesperidin together form at least 50% of thebioflavonoid content.
 3. The material of claim 2, wherein the naringinand neohesperidin together form at least 70% of the bioflavonoidcontent.
 4. The material of claim 3, wherein the naringin andneohesperidin together form at least 75% of the bioflavonoid content. 5.The material of claim 4, wherein the naringin and neohesperidin togetherform between 75% and 80% of the bioflavonoid content.
 6. The material ofany preceding claim, wherein the bioflavonoid content of the compositionfurther comprises one or more compounds selected from the group ofneoeriocitrin, isonaringin, hesperidin, neodiosmin, naringenin, poncirinand rhiofolin.
 7. The material of claim 6, wherein the bioflavonoidcontent of the composition comprises neoeriocitrin, isonaringin,hesperidin, neodiosmin, naringenin, poncirin and rhiofolin.
 8. Thematerial of any preceding claim, wherein the bioflavonoid composition isuniform throughout the material.
 9. The material of any preceding claim,wherein the material is paper.
 10. The material of any preceding claim,wherein the material is bamboo fibre.
 11. The material of either ofclaim 9 or 10, wherein the paper or bamboo is in the form of a towel ora cloth.
 12. The material of either of claim 9 or 10, wherein the paperor bamboo is in the form of a food pad.
 13. The material of either ofclaim 9 or 10, wherein the paper or bamboo is in the form of arespiratory mask.
 14. The material of claim 10, wherein the bamboo is inthe form of a garment such as a sock or hospital gown.
 15. The materialof any of claims 1 to 8, wherein the material is cardboard.
 16. Aprocess for impregnating a cellulosic fibrous material with abioflavonoid composition comprising the steps of: a) immersing orspraying the material or material fibres with the bioflavonoidcomposition defined in any of claims 1 to 7; b) mechanically drying thematerial.
 17. A method of reducing the bacterial load on a surfacecomprising the steps of: a) wetting the material defined in any of claim9, 10 or 11 with moisture; and b) placing the material on a surfaceand/or wiping the material across the surface.
 18. The method of claim17, wherein the bacterial load is comprised of bacteria selected fromthe group of S. aureus, E. coli, P. aeruginosa, B. subtilis, Salmonella,MRSA, C. diff and H. pylori.
 19. A packaged product comprising a drycellulosic fibrous material as defined in any of claims 1 to
 8. 20. Theproduct of claim 19, wherein cellulosic fibrous material is as definedin any of claim 9, 10 or
 15. 21. The product of either of claim 19 or20, wherein the product is in the form of a towel or cloth.
 22. Theproduct of either of claim 19 or 20, wherein the product is in the formof a food pad.
 23. The product of either of claim 19 or 20, wherein theproduct is in the form of a respiratory mask.
 24. The product of eitherof claim 19 or 20, wherein the product is in the form of a garment suchas a sock.